Hundreds of millions of people are infected with Dengue and Zika viruses (DENV and ZIKV), flaviviruses that can lead to life-threatening diseases (DENV) or devastating congenital abnormalities such as microcephaly (ZIKV). Unfortunately, no antiviral treatments are available to combat either virus, and efforts in developing antiviral therapeutics have been hampered by a paucity of information on critical virus-host interactions that regulate replication and fitness of these viruses. Mammalian cells are equipped with elaborate, intrinsic mechanisms to fend off invading viruses, but little is known about host factors that restrict DENV or ZIKV. Our recent in vitro studies have shown that TRIM56, a tripartite-motif protein, is a novel antiviral host factor that possesses E3 ubiquitin ligase activity to restrict DENV, ZIKV, and two closely related viruses, yellow fever virus and bovine viral diarrhea virus. Yet, the underlying mechanism remains elusive, as does the physiologic role of TRIM56 in flavivirus infection in vivo. In consideration of the broad antiviral activity of TRIM56 against four different flaviviruses (BVDV, YFV, DENV and ZIKV) that have limited homology in viral proteins, we hypothesize that the TRIM56 E3 ligase targets a (subset of) host factor(s) that has common, regulatory role(s) in replication and/or fitness of different flaviviruses, a better understanding of which may inform antiviral strategies that have broad activity against medically important flaviviruses including DENV and ZIKV. Two independent specific aims are proposed in this application: In Aim 1, we will determine the molecular mechanisms underlying the anti-flavivirus action of TRIM56. Specifically, we will address the roles of several host-encoded RNA-binding proteins, which we found TRIM56 forms a complex with, in DENV and ZIKV RNA replication and determine their regulation by TRIM56. In Aim 2, we will determine the role of TRIM56 in host antiviral defense against ZIKV infection in vivo, using a TRIM56 knockout mouse model we recently developed. Results from the proposed studies will yield novel insights into host factors that regulate DENV and ZIKV replication and fill key knowledge gaps concerning the antiviral mechanism and precise role of TRIM56 in flavivirus restriction. The gained new knowledge may inform the development of antiviral therapeutics against Dengue, Zika and possibly other flavivirus infections.